The goal of the proposed research is to achieve in-depth understanding of the molecular mechanisms underlying the CNS pathogenesis in GM1-gangliosidosis, a catastrophic neurodegenerative lysosomal storage disease (LSD) caused by deficiency of 2-galactosidase (2-gal). In this disease massive and progressive accumulation of GM1-ganglioside (GM1) particularly in the CNS is thought to play a major role in disease pathogenesis, albeit the molecular pathways involved remain largely unexplored. Gangliosides are sialic acid- containing glycosphingolipids (GSLs) particularly abundant in the nervous system. As key modulators of intracellular Ca2+ flux, they have been implicated in cellular processes downstream of Ca2+ signaling. Changes in their chemical composition and concentration can therefore alter normal cell function and lead to cell death. Our successful generation of a 2-gal-/- mouse model that closely resembles the human disease has enabled us to initiate a comprehensive analysis of the molecular bases of neurodegeneration characteristic of this disorder in children. We have demonstrated that in these mice excessive intracellular concentration of GM1 induces apoptosis by depletion of endoplasmic reticulum (ER) Ca2+ stores and activation of an unfolded protein response (UPR). Given the spatial interplay between the ER and the mitochondria and the role of Ca2+ in conveying apoptotic signals emanating from these compartments, we now plan to investigate whether GM1 can induce both ER stress- and mitochondria-mediated apoptosis by influencing Ca2+ homeostasis in these organelles. The studies put forward in the two Aims of this proposal are designed to elucidate the following points. 1) We will examine the potential effect(s) of increased levels of GM1 on ER membrane channels and pump that regulate intracellular Ca2+ concentration. 2) We will focus on the role of GM1 on specific membrane components of the mitochondria-associated membranes or MAMs, the sites of juxtaposition between ER and mitochondrial membranes that regulate the kinetics of Ca2+ flux between these organelles. 3) We will study the downstream effects of ER Ca2+ depletion on the mitochondria. Considering the role of the Bcl-2 family of proteins in both ER- and mitochondria-mediated apoptosis, and their response to Ca2+ levels in these organelles, 4) we will explore a putative, additional involvement of these proteins in the neurodegenerative events characteristic of GM1-gangliosidosis. PUBLIC HEALTH RELEVANCE: GM1-gangliosidosis is a catastrophic neurodegenerative disease that affects infants and children for which there is no treatment. We are in the position to gain full understanding of the events in this disease that cause cell death in the brain. We are confident that these studies will enable the identification of novel mechanisms of pathogenesis and may reveal basic biological processes controlled by GM1. This knowledge is essential for designing new therapies for children with GM1-gangliosidosis and possibly those with other GSL storage diseases.